Selectivity Optimization in Tandem Column Liquid Chromatography Using the Eluent Composition as the Tuning Variable

Abstract

Clearly, in typical separations in HPLC, selectivity is the most important variable for improving resolution and the optimization of selectivity is the primary focus of most method development effort in reversed-phase liquid chromatography (RPLC). We have developed a family of novel techniques in which two columns with markedly different chromatographic selectivities are combined in tandem. One then uses another chromatographic variable (e.g. temperature, flow rate, eluent composition) which has different values for the two columns to continuously “tune” the selectivity. We call this family of techniques XT2C, meaning that the variable X (temperature, etc.) is used to continuously tune the combined selectivity of the tandem columns. By serially coupling the columns, our approach eliminates the common “selectivity discontinuity” problem encountered by analysts when the type of column used is changed. Previously, we utilized the thermally tuned tandem column (T3C) concept in which selectivity was adjusted by independently tuning the two column temperatures. The primary difference between the experimental setup for T3C and XT2C is that a second pump and static micro-mixer are placed between the two columns. In this study, we describe the eluent tuned tandem column concept in which selectivity is continuously tuned by independently controlling the eluent composition of each column. When the percent organic modifier in the eluent (%B) is adjusted, the concept is denoted “solvent tuned tandem column (ST2C)”. Likewise, when the ionic strength (buffer concentration) is tuned, the concept is referred to as ionic strength tuned tandem column (IT2C). The objective of the present study is to evaluate the applicability of IT2C and ST2C to mixtures of ionic and non-ionic analytes, respectively. We also describe the use of a simple computer assisted optimization strategy based on the window diagram method. This strategy allows for XT2C optimization based on only four to six initial data acquisition runs. ST2C and IT2C are comparable in terms of chromatographic performance to T3C and provide flexibility in optimizing selectivity but do not require thermally stable columns.